Since the original description of adipose tissue as a source of adult multilineage stem cells [1
], the preclinical study of adipose-derived cells for repair and regeneration of tissues has increased dramatically (reviewed in [2
]). The theoretical advantages of adipose-derived cells over other cell types for biologic bone regeneration strategies are numerous. For example, although autograft bone remains the gold standard for bone repair, its disadvantages are significant, including donor site morbidity, extended operating time complications, and limited autogenous supply [3
]. On the other hand, bone marrow mesenchymal stem cells (BMSCs) have long been studied for their application to skeletal engineering. However, BMSCs have distinct disadvantages over adipose-derived cells, including limitations in autogenous supply of bone marrow aspirate as compared with lipoaspirate, and relatively lower cell yield of BMSCs in comparison with adipose-derived stem cells (ASCs) (estimated at ~104
stem cells vs. ~106
stem cells per 40 ml) [6
]. Finally, human ASCs have been used successfully to heal small animal [9
] and large animal [11
] skeletal defects.
Despite the advantages of fat tissue-derived cells for bone regeneration, there exist clear hurdles that must be overcome for their successful clinical use. For example, adipose-derived stromal cells are defined by their adherence to cell culture plates and ex vivo expansion. This ex vivo expansion increases the risk of immunogenicity, infection, and genetic instability [12
]. As an alternative to ASCs, the cells of noncultured total stromal vascular fraction (SVF) from adipose tissues have been used. Available studies using SVF show poor and unreliable bone formation [14
] or lower bone regeneration efficacy relative to cultured ASCs [15
]. SVF of adipose tissue is a highly heterogeneous cell population, including nonmesenchymal stem cell types, such as inflammatory cells, hematopoietic stem cells, and endothelial cells, among others [16
]. Variability in cell composition presents clear disadvantages for U.S. Food and Drug Administration (FDA) approval of a future stem cell-based therapeutic, potentially including reduced safety, purity, identity, potency, and efficacy. Notably, these are the criteria upon which the Center for Biologics Evaluation and Research evaluates stem cell-based product applications [17
]. For example, given the heterogeneity of SVF, precise product characterization is not feasible, leading to lack of human stromal vascular fraction (hSVF) product identity. Batch-to-batch variability and nonuniformity in effect (again attributable to variable stem cell content) reduces the efficacy and potency of hSVF compounds and potentially reduces product safety. With these regulatory hurdles in mind, our approach was to reduce the inherent heterogeneity within hSVF to obtain a safer and more efficacious stem cell-based therapeutic.
To address this issue of SVF heterogeneity, previous investigators have used fluorescence-activated cell sorting (FACS) to purify fat-derived cell populations, with some success. For example, cell sorting of adipose tissue has allowed for enrichment of osteoprogenitor [18
] or chondroprogenitor [19
] cell types—but not the isolation of multipotent mesenchymal stem cells (MSCs). Our solution has been to identify the native perivascular phenotype of MSCs and prospectively isolate perivascular MSCs using multicolor FACS. We have previously shown two populations of perivascular stem cells (PSCs) associated with blood vessels [20
]. These include CD34−CD146+CD45− pericytes surrounding microvessels and capillaries [20
] and a second distinct adventitial cell (CD34+CD146−CD45−) [26
], associated with larger blood vessels. Both cell types are isolatable from adipose tissue and resemble MSCs by morphology, growth, surface markers, and clonal multilineage differentiation potential [20
In this report, we describe the use of a novel, FACS-purified cell source for improved bone tissue engineering: human perivascular stem cells (hPSCs). Using an intramuscular, ectopic bone model, we observed that adipose-derived, FACS-isolated hPSCs formed bone to a greater degree than patient-matched, unsorted hSVF. Next, two growth factors were examined for their osteoinductive effects: bone morphogenetic protein 2 (BMP2) and Nel-like molecule-1 (NELL-1). The novel cytokine NELL-1 was identified as an appropriate osteogenic stimulus rather than BMP2, which had pleiotropic effects, including adipogenic differentiation and cyst-like bone formation.